CN108588117B - Application of Qinghai-Tibet plateau wild barley HsCIPK17 in improving abiotic stress resistance of rice - Google Patents
Application of Qinghai-Tibet plateau wild barley HsCIPK17 in improving abiotic stress resistance of rice Download PDFInfo
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Abstract
The invention discloses an application of a calcineurin B protein interaction protein kinase gene HsCIPK17 of annual wild barley (Hordeum spontanemum C. Koch) in Qinghai-Tibet plateau: HsCIPK17 is used for constructing transgenic rice (Oryza sativa L.), and the transgenic rice has heavy metal stress tolerance and also has salt tolerance and abscisic acid stress tolerance; the heavy metal is mercury, cadmium or chromium; the nucleotide sequence of the gene HsCIPK17, GenBank accession number JN 655677.
Description
Technical Field
The invention belongs to the field of plant genetic engineering. Specifically, the invention relates to application of wild barley HsCIPK17 in Qinghai-Tibet plateau in improving abiotic stress resistance of rice.
Background
In order to adapt to the changing environment, plants must cope with various biotic and abiotic stresses, and thus a series of regulatory mechanisms are established during evolution, such as sensing and decoding of various stress signalsSignal transduction and expression of stress-related genes. During the growth and development of plants, Ca2+The signal is a central regulator of the physiological response to plant cell stress (Dodd et al, 2010). Ca2+Changes in signal are first sensed and decoded by calcineurs, which in turn transmit the signal downstream by calcineurin-interacting proteins, thereby activating expression of downstream early response genes, ultimately leading to a wide range of physiological or a variety of specific stress responses (Zhu et al, 2013). In the past decade, calcineurin B-like protein (CBL), a calcineurin, and CBL-interacting protein kinases (CIPKs), are Ca-like proteins2+Dependent serine/threonine kinases have been shown to be a plant specific signaling system that plays a role in signaling functions in plants in response to various biotic and abiotic stresses (Kolukissaoglu et al, 2004; Xu et al, 2006; Luan 2009; Weinl and Kudla 2009; Hashimoto and Kudla, 2011; de la Torre et al, 2013).
CIPKs play an important role in various biotic stresses (such as pathogen infection, nutritional deficiencies, etc.) and abiotic stresses (such as salt, abscisic acid (ABA), etc.) (Guo et al, 2001; Kim et al, 2003; Kurusu et al, 2010; Li et al, 2012; de la Torre et al, 2013; meteigner et al, 2017). In rice (Oryza sativa L.), overexpression of OsCIPK3, OsCIPK12 and OsCIPK15 can improve the resistance to drought, low temperature and salt stress (Xiaong et al, 2007), while deletion of a functional gene mutant of OsCIPK31 shows sensitivity to various abiotic stresses such as abscisic acid (ABA), salt, mannitol and glucose (Piao et al, 2010) in seed germination and seedling stages, but there is no evidence to date that CIPK participates in studies related to heavy metal toxicity responses of plants.
Qinghai-Tibet plateau wild barley, especially its characteristic annual wild barley (Hordeum spontanemum C. Koch), is subjected to extreme climatic and environmental conditions for long periods of time, resulting in abundant variants and a unique network of stress tolerance genes. Since wild barley of the Qinghai-Tibet plateau has close genetic homology with cultivated barley, the Qinghai-Tibet plateau is considered as one of the centers for domestication of cultivated barley (Dai et al, 2012). CIPK (clean in place) of wild barley in Qinghai-Tibet plateau is a scarce germplasm resource, and has important significance for improving the capability of adversity stress of transgenic rice or other crops.
Abscisic acid (ABA) participates in important physiological processes of plants and responses to various adversity stresses, and the existing research shows that Arabidopsis AtCIPKs participate in regulating ABA-mediated signal pathways (Chen L et al, 2013; Lyzenga et al, 2013; Sanyal et al, 2017). Heavy metals are important stress factors for plant growth and harm animals and humans through the food chain (yao et al, 2013; castration et al, 2017). Because heavy metals are extremely stable and non-degradable, contaminated soil causes irreversible damage to plant growth and development and crop yield and quality (musafa and Komatsu, 2016). It is the best solution to improve the high resistance of crops to heavy metal poisoning by genetic engineering (Cao et al, 2014).
At present, no CIPKs genes are known to have abiotic stress resistance/tolerance to heavy metals and salts.
The above references are specifically as follows:
1. cao FB, Wang RF, Cheng WD, Zeng FR, Ahmed IM, Hu XN, Zhang, GP, and Wu FB (2014) genomic and environmental variation in calcium, chromium, lead and copper in and with additives for reducing the accumulation of the Science of the Total Environment,496: 275-;
2. chen L, Wang QQ, Zhou L, Ren F, Li DD, Li XB (2013) Arabidopsis CBL-interacting protein kinase (CIPK6) is involved in plant response to salt/osmotic stress and ABA. Mol Biol Rep.40:475947-67(Chen L, Wang QQ, Zhou L, Ren F, Li DD, Li XB (2013) Arabidopsis CBL-interacting protein kinase (CIPK6) is involved in plant response to salt/osmotic stress and ABA. Mol Biol Rep.40: 475947-67);
3. dai F, Nevo E, Wu DZ, Comadran J, Zhou MX, Qiu L, Cheng ZH, Belles A, Chen GX, Zhang GP (2012) Tibet is one of the centers of the society of clinical barley. Proc Natl Acad Sci USA 109: 16969-;
4. de la Torre F, Guti rrez-Beltr-n E, Pareja-Jaime Y, Chakravarty S, Martin GB, del Pozo O (2013) The tomato calcum sensor Cbl10and its interacting protein kinase Cipk6define a signalling pathway in Plant immunity Plant cell.25:2748-2764(de la Torre F, Guti rrez-Beltr-n E, Pareja-Jaime Y, Chakravarty S, Martin GB, del Pozo O (2013) tomato calcium receptor Cbl10and its interoperable protein kinase Cipk6 are a signalling pathway of Plant immunity cell 2748);
5. dodd AN, Kudla J, Sanders D (2010) The language of calcium signalling, Annu Rev Plant biol.61: 593-;
6. guo Y, Hall U, Ishitani M, and Zhu JK (2001) Molecular characterization of functional domains in the protein kinase SOS2that is required for Plant tolerance. Plant Cell,13: 1383-;
7. hashimoto K, Kudla J (2011) Calcium decoding mechanisms in plants Biochimie.93:2054-2059(Hashimoto K, Kudla J (2011) decoding mechanism for Calcium in plants Biochimie.93: 2054-2059);
8. kim KN, Cheng YH, Grant JJ, Pandey GK, and Luan S (2003) CIPK3, A calcium sensor-associated protein kinase that regulates abscisic acid and color signal transduction in Arabidopsis Cell,15:411-423(Kim KN, Cheng YH, Grant JJ, Pandey GK, and Luan S (2003) Arabidopsis thaliana calcium-related protein CIPK3 regulates abscisic acid and cold signal transduction calcium. Plant Cell,15: 411-423);
9. kolukisaloglu U, Weinl S, Blazevic D, Batistac O, Kudla J (2004) Calcium sensors and the same interacting proteins enzymes, genetics of the Arabidopsis and rice CBL-CIPK signaling networks Plant Physiol.134:43-58 (Kolukisaloglu U, Weinl S, Blazevic D, Batistac O, Kudla J (2004) Calcium Arabidopsis and its interactor kinase: and the genomics of the rice CBL-CIPK signal network Plant Physiol.134: 43-58);
10、Kurusu T,Hamada J,Nokajima H,Kitagawa Y,Kiyoduka M,Takahashi A,Hanamata S,Ohno R,Hayashi T,Okada K et al.(2010)Regulation of microbe-associated molecular pattern-induced hypersensitive cell death,phytoalexin production,and defense gene expression by calcineurin B-like protein-interacting protein kinases,OsCIPK14/15plant Physiol,153:678-692(Kurusu T, Hamada J, Nokajima H, Kitagawa Y, Kiyoduka M, Takahashi A, Hanamata S, Ohno R, Hayashi T, Okada K et al (2010) calcineurin B-like protein-interacting protein kinase OsCIPK K in cultured cells of rice14/15Regulating and controlling the relevant molecule mode of the microorganism to induce hypersensitive cell death, phytoalexin generation and defensive gene expression Plant Physiol,153: 678-692);
11. li R, Zhang J, Wu G, Wang H, Chen Y, and Wei J (2012) HbCIPK2, a novel CBL-interacting protein kinase from halophthalate Hordeum brevuluuctum, conjugates salt and osmotic stress tolerance Plant Cell Environ,35: 1582-;
12. luan S (2009) The CBL-CIPK network in Plant calcium signaling. Trends Plant Sci.14:37-42(Luan S (2009) CBL-CIPK network for Plant calcium signaling. Trends Plant Sci.14: 37-42);
13. lyzenga WJ, Liu H, Schofield A, Muise-Hennessey A, Stone S (2013) Arabidopsis CIPK26 interactions with KEG, compositions of the ABA signalling network and is degraded by the ubiquitin-protease system.J Exp Bot,64(10) 2779-;
14. meteignier LV, El Oirdi M, Cohen M, Barff T, Matteau D, Lucier JF, Rodrigue S, Jacques PE, Yoshioka K, and Moffett P (2017) translometers analysis of an NB-LRR immune responses to Arabidopsis in Arabidopsis Bop, 68:2333-2344(Meteignier LV, El Oirdi M, Cohen M, Barff T, Matteau D, Lucier JF, Rodrigue S, Jacques PE, Yoshioka K, and Moffett P. (2017) Arabidopsis thaliana NB-R translation component demonstrating important effects on plant immunity J. 2344. LR.14p.2333-2334);
16、Mustafa G and Komatsu S(2016)Toxicity of heavy metals and metal-containing nanoparticles on plants.Biochim Biophys Acta,1864:932-494(Mustafa G and Komatsu S(2016)
toxicity of heavy metals and metal nanoparticles to plants Biochim Biophys Acta,1864: 932-494);
17. piao HL, Xuan YL, Park SH, Je BI, Park SJ, Park SH, Kim CM, Huang J, Wang GK, Kim MJ (2010) OsCIPK31, a CBL-interacting protein kinase is involved in formation and seed growth under abiotic stress conditions in rice seed germination and seedling growth in rice seed cell 30:19-27(Piao HL, Xuan YL, Park SH, Je BI, Park SJ, Park SH, Kim CM, Huang J, WaGK, Kim MJ (2010) under abiotic stress CBL interacting protein kinase OsCIPK31 participates in rice seed germination and seedling growth in rice seed cell 30: 19-27);
18. sanyal SK, Kanwarp, Yadav AK, Sharma C, KumarA, Pandey GK (2017) Arabidopsis CBL interacting protein kinase 3 interactions with ABR1, an APETALA2domain transcription factor, to regulated ABA responses Plant Sci,254:48-59(Sanyal SK, Kanwarp, Yadav AK, Sharma C, KumarA, Pandey GK (2017) Arabidopsis thaliana CBL interacting protein kinase 3interacts with the ABR1 domain transcription factor ABR 2 to modulate ABA responses Plant Sci,254: 48-59);
19、Weinl S,Kudla J(2009)The CBL-CIPK Ca2+-decoding signaling network:function and perspectives.New Phytol.184:517-528(Weinl S,Kudla J(2009)CBL-CIPK Ca2+ encoded signal network function and perspective New phytol.184: 517-528);
20. xiaong Y, Huang Y, Xiong L (2007) Characterization of stress-responsive CIPK genes in rice for stress tolerance improvement. Plant physiology.144: 1416-;
21、Xu J,Li HD,Chen LQ,Wang Y,Liu LL,He L,Wu WH(2006)A protein kinase,interacting with two calcineurin B-like proteins,regulates K+transporter AKT1in Arabidopsis cell 125:1347-1360(Xu J, Li HD, Chen LQ, Wang Y, Liu LL, He L, Wu WH (2006) protein kinase regulation Arabidopsis K + transporter Akt1 cell 125:1347-1360) interacting with two calcineurin B-like proteins;
22、Zhu S,Zhou X,Wu X,Jiang Z(2013)Structure and function of the CBL-CIPK Ca2+CBL-CIPK Ca in coding system in plant calcium signalling plant Mol Biol Rep.31:1193-1202(Zhu S, Zhou X, Wu X, Jiang Z (2013) plants2+The structure and function of the decoding system Plant Mol Biol Rep.31: 1193-);
23. joule position male, Yangherde, von Dene, forest pine, Li Chong Campsis (2017) research on heavy metal content and accumulation characteristics of edible parts of different crops under Cd Hg Pb stress, agricultural environmental science, 36(9):1726 + 1733;
24. the influence of heavy metal pollution on the quality and safety of agricultural products by the gayao, the Li Steel, the Fei Fange, the Cao Qingjun and the Populus starch ball (2013) and the prevention and treatment measures thereof, wherein the quality and the safety of the agricultural products are 21(3) to 9-14.
Disclosure of Invention
The invention aims to solve the technical problem of providing the application of the Qinghai-Tibet plateau wild barley HsCIPK17 in improving the abiotic stress resistance of rice, and the obtained transgenic plant has abiotic stress tolerance of heavy metal, salt, abscisic acid (ABA) and the like.
In order to solve the technical problems, the invention provides the application of a calcineurin B protein interacting protein kinase (CBL-interacting protein kinase, CIPK) gene HsCIPK17 of annual wild barley (Hordeum spontanenum C. koch) in Qinghai-Tibet plateau: HsCIPK17 is used for constructing transgenic rice (Oryza sativa L.) which has heavy metal stress tolerance;
the nucleotide sequence of the gene HsCIPK17, GenBank accession number JN 655677.
As an improvement of the use of the wild barley gene HsCIPK17 of the present invention: the transgenic rice has salt tolerance and abscisic acid (ABA) stress tolerance at the same time.
Namely, the transgenic rice HsCIPK17 has 3 abiotic stress tolerance of heavy metal, salt tolerance and abscisic acid (ABA) at the same time.
As a further improvement of the use of the wild barley gene HsCIPK17 of the invention: the heavy metal is mercury, cadmium or chromium.
Namely, the transgenic rice HsCIPK17 has mercury resistance, cadmium resistance and chromium resistance; also has salt resistance and abscisic acid (ABA) resistance.
As a further improvement of the use of the wild barley gene HsCIPK17 of the invention: the rice mature embryo callus is transformed by the HsCIPK17 gene, and then the transformed rice cells are cultivated into a transgenic plant which has 3 abiotic stress tolerance of heavy metal, salt tolerance and abscisic acid (ABA).
The specific technical steps for realizing the invention are as follows:
cloning of HsCIPK17 gene of annual wild barley in Qinghai-Tibet plateau
The wild barley HsCIPK17 gene is cloned by a PCR technology, and the HsCIPK17 gene coding sequence CDS is constructed to a cauliflower mosaic virus 35S promoter vector by a molecular biology technology such as enzyme digestion and connection. See figures 1, 2 and table 1.
Second, rice transgenosis
The 35S-HsCIPK 17 expression vector is introduced into rhizobium allelopathic state (EHA105) by an electric shock method, rice Nipponbare mature embryo callus is infected by agrobacterium, and a transgenic plant with HsCIPK17 gene overexpression is obtained by screening and differentiating resistant callus. See fig. 3 and table 2.
Expression of exogenous HsCIPK17 gene in rice transgenic plant
The exogenous HsCIPK17 gene is introduced into rice by transgenic technology, and the expression level of the exogenous HsCIPK17 gene is detected in a T1 transgenic plant by using methods such as semi-quantitative RT-PCR and the like, so that an over-expressed transgenic rice plant is finally obtained. See fig. 4 and table 1.
Screening of T3 transgenic homozygous lines
After the high-expression strain is propagated to T3 generation, screening homozygous strain, and judging homozygote or heterozygote according to the segregation ratio. See fig. 5 and table 2.
Five, heavy metal and other abiotic stresses induce the transcription expression of endogenous HsCIPK17 of the annual wild barley
Detection of heavy metal salts (HgCl) by real-time quantitative PCR (qRT-PCR)2、CdCl2And K2Cr2O7) And the effects of other abiotic stresses (salt and ABA) on the transcriptional expression levels of endogenous hscpk 17 in the annual wild barley from tibetan plateau. See fig. 6 and table 1. Respectively with 20 μ M HgCl2(FIG. 6A), 20. mu.M CdCl2(FIG. 6B), 0.5mM K2Cr2O7The results of testing the transcriptional expression of endogenous HsCIPK17 after 1, 3, 6, 12 and 18 hours of stress treatment (FIG. 6C), 400mM NaCl (FIG. 6D) and 20. mu.M ABA (FIG. 6E) indicated that the expression level was significantly increased at different times of each stress.
Six, T3 transgenic homozygous strain HsCIPK17 gene function identification
Selecting T3 transgenic homozygous line rice seeds and non-transgenic seeds, accelerating germination, and culturing seedlings for 4 days for stress treatment of heavy metal, salt, ABA and the like. 0.5. mu.M HgCl2(FIG. 7A), 5. mu.M CdCl2(FIG. 7B), 5. mu. M K2Cr2O7(FIG. 7C), NaCl 50mM (FIG. 7D) and 1. mu.M ABA (FIG. 7E), blank treatment was 0.1mM CaCl2After 24 hours of treatment, the elongation of the primary root is measured and the relative elongation of the primary root is calculated as RER (%). The result shows that the HsCIPK17 transgenic rice plant has obvious resistance/tolerance to abiotic stress treatment such as heavy metal, salt, ABA and the like (figure 7).
Due to the acceleration of industrialization, the heavy metal pollution of cultivated land is serious, and the statistics of the ministry of national resources shows that more than 10 percent of the area of cultivated land in China is polluted by heavy metal. In order to fully utilize the cultivated lands, the cultivation of crop varieties with strong tolerance resistance is urgently needed, and the development of the genetic engineering technology makes the application of CIPK gene to adjust crops to adapt to different heavy metal stress conditions possible. The invention obtains the HsCIPK17 gene of the annual wild barley in Qinghai-Tibet plateau by a cloning technology, obtains an over-expression material by transgenosis, and preliminarily identifies the function of the gene.
The invention makes the rice have good stress tolerance such as heavy metal resistance, thereby promoting the growth and development of the rice. The invention defines the functions of HsCIPK17 in the aspects of heavy metal toxicity resistance, salt tolerance, abscisic acid (ABA) and the like, and the invention results can be used for regulating the expression of CBL interacting protein kinase genes of crops through biotechnology, thereby cultivating new varieties of crops with obviously improved environmental stress resistance or yield. The invention has good application prospect.
In conclusion, the invention relates to a gene HsCIPK17 of calponeurin B-like protein kinase (CBLs) interacting protein kinases (CIPs) of annual wild barley (Hordeum spontanenum C. Koch) of Qinghai-Tibet plateau through PCR cloning, and HsCIPK17 overexpression rice plants are obtained through a transgenic technology; the gene is also used for regulating and controlling the resistance/tolerance of the rice under heavy metal adversity stress and other abiotic stress, thereby enhancing the growth and development of the rice under the adversity condition and improving the yield of the rice.
The invention successfully clones the HsCIPK17 gene from the annual wild barley in Qinghai-Tibet plateau by a molecular cloning technology, constructs a HsCIPK17 overexpression vector, introduces the HsCIPK17 expression vector into a Nipponbare rice variety by utilizing an agrobacterium tumefaciens mediated rice genetic transformation technology, screens and identifies transgenic offspring, and breeds a high-expression transgenic plant line. The invention discovers that HsCIPK17 can obviously improve the resistance/tolerance capability of transgenic rice plants to abiotic stresses such as heavy metals (such as mercury, cadmium and chromium), salt and ABA.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 is a schematic diagram of a rice transformation overexpression vector;
a is a simple structure schematic diagram; b is a plant expression vector map;
2 x 35S is cauliflower mosaic virus 35S promoter vector (pCAMBIA2300-2 x 35S), the vector has no gus reporter gene and contains Kanamycin (Kanamycin) resistance gene, the 35S promoter is followed by full-length HsCIPK17cDNA, and the transgenic plant of over-expression CIPK17 can be obtained after the transgenic use.
FIG. 2 is a diagram of the construction of the HsCIPK17 overexpression vector;
(A) HsCIPK17PCR product;
(B) performing PCR positive clone identification, wherein CK is a control, 1-23 represent different strains respectively, and 19 and 22 represent two positive clone strains;
(C)35S, double enzyme digestion identification of HsCIPK 17; m: DNA molecular weight markers; d: enzyme digestion; u: no digestion was performed.
FIG. 3 shows transgenic seedlings obtained by genetic transformation of rice;
(A) callus induction, namely inducing the rice Nipponbare mature embryo to callus on an N6D culture medium for transgenosis;
(B) co-culturing, namely introducing agrobacterium EHA105 of an overexpression vector of HsCIPK17, infecting callus, and performing dark culture on a co-culture medium;
(C) resistance callus screening, transferring the callus to a culture medium containing G418 after bacteria removal for resistance screening (because rice has higher resistance to kanamycin), and growing new resistance callus;
(D and E) callus differentiation, transferring the resistant callus to a differentiation culture medium for induction differentiation, and growing into a plantlet;
(F) rooting culture, transferring the differentiated plantlets to a rooting culture medium, and growing roots from the base parts of the plantlets;
(G.) hardening seedlings, and transplanting the seedlings into a field for planting after hardening seedlings.
FIG. 4 shows the expression level of the exogenous HsCIPK17 gene identified by semi-quantitative RT-PCR in transgenic rice plants of the T1 generation;
transferring 35S, namely HsCIPK17 strain, NC: negative control strain nipponica; OsActin is an internal reference; the numbers are numbers of different strains; and 1-9 are the serial numbers of the strains.
FIG. 5 is T3 generation homozygous line screen;
(A) g418 resistant culture medium screening, left homozygote strain and right wild strain;
(B) the growth of the roots on the medium, the left resistant strain, the right wild type strain, was selected.
FIG. 6 shows the transcription and expression result of endogenous HsCIPK17 of the annual wild barley induced by abiotic stress such as heavy metal;
total RNA of seedlings or roots of 4-day-old annual wild barley seedlings treated with heavy metal and other abiotic stresses for 0, 1, 3, 6, 12 and 18 hours were extracted with RNeasy Plant Mini Kit (Qiagen) RNA Kit and measured. Relative expression levels are expressed as relative mean ± standard deviation, with 0 hour treatment group as control, and by t-test, P < 0.01, 0.001 and 0.0001 are expressed, respectively.
FIG. 6A 20. mu.M HgCl2Treatment, relative expression level of hscpk 17;
FIG. 6B 20. mu.M CdCl2Treatment, relative expression level of hscpk 17;
FIG. 6C 0.5mM K2Cr2O7Treatment, relative expression level of hscpk 17;
FIG. 6D 400mM NaCl treatment, relative expression levels of HsCIPK 17;
figure 6E relative expression levels of HsCIPK17 for 20 μ M ABA treatment.
FIG. 7 shows the results of identifying heavy metals and other abiotic stresses in rice plants overexpressing HsCIPK 17;
t3 transgenic homozygous strain rice seed and non-transgenic (NT) seed, seedling culture to root length 3-4cm, after carrying on abiotic stress treatment such as heavy metal, salt and ABA separately for 24 hours, determine the elongation of the primary root, and calculate the relative elongation RER (%) of the primary root, the result is expressed as relative mean value + -standard deviation, use NT as the control, through T test, expression P < 0.01 and 0.001 separately;
FIG. 7A 0.5. mu.M HgCl2FIG. 7B 5. mu.M CdCl2FIG. 7C 5. mu. M K2Cr2O7FIG. 7D 50mM NaCl, FIG. 7E 1. mu.M ABA.
Detailed Description
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
example 1 seed Germination and culture
Seeds of the annual wild barley X74(Hordeum spontanemum c. koch) and the japanese sunny rice (Oryza sativa l. ssp. japonica) in the Qinghai-Tibet plateau were surface-sterilized with 70% ethanol for 10 minutes, then sterilized with 10% NaClO for 30 minutes, and finally rinsed 8 times with water. Barley and rice grain endosperm contains a large amount of nutrients sufficient to meet the nutritional needs for grain germination within a week. Thus, a simple CaCl2Solution (0.1mM CaCl)2(ii) a pH 5.8) for barley and rice seed germination and seedling growth. Placing the sterile barley seeds in CaCl at 25 deg.C in dark2Germinating between two layers of filter paper soaked in the solution for one day, transferring the seeds to new CaCl2The solution-soaked absorbent cotton filter paper was incubated for a further 4 days (dark 25 ℃). Treating rice seeds in the dark (4 deg.C) for 3 days, germinating at 37 deg.C for 3 days, and transferring germinated seeds to CaCl2The solution soaked absorbent cotton filter paper was incubated for a further 4 days (14 hours light 28 ℃ C./10 hours dark 25 ℃ C.). Barley and rice seedlings of 4 days old with similar root lengths were used for the study of the present invention.
Example 2 cloning of the HsCIPK17 Gene of annual wild barley in Qinghai-Tibet plateau
(1) Two methods were used to electronically clone HsCIPK 17. First, known rice plants OsCIPK1 to OsCIPK31(http://www.ncbi.nlm.nih.gov/(ii) a OsCIPK cDNAs gene accession numbers are as follows) full-length cDNAs are used as probes, and homologous genes are searched in a barley (Hordeum vulgare L.) full-length cDNA library (homology gene)http://earth.lab.nig.ac.jp/～ dclust/cgi-bin/barley_pub/). Secondly, conserved sequence activation loops and NAF/FISL motifs in full-length cDNAs of rice OsCIPK1 to OsCIPK31 as probes and barley nucleotide collection (nr/nt) database (r/nt)http://blast.ncbi.nlm.nih.gov/) BLAST alignment of homologous fragment sequences, electronic splicing and extension. Finally, the resulting full-length cDNA was analyzed with DNA STAR SeqMan and Megalign software.
OsCIPK1-OsCIPK31cDNAs NICB Genbank accession number
(2) Total RNA was extracted from 4-day-old seedlings by the method described in the general RNA extraction Kit RNeasy Plant Mini Kit (QIAGEN). OD was measured using NanoDrop. The total RNA obtained above is used for cDNA synthesis, and the synthesis method is shown in a reverse transcription kit
III Reverse Transcriptase (Invitrogen) manufacturer.
Remarks explanation: gene HsCIPK17, GenBank accession JN 655677; the nucleotide sequence is shown in SEQ ID NO. 1.
Example 3 transgenic 35S of HsCIPK17 Gene construction of HsCIPK17 vector
35S (HsCIPK 17) is constructed by PCR, restriction enzyme cutting and splicing with a plant transformation vector pCAMBIA2300S (figure 1). pCAMBIA2300S contains the 2 × CaMV 35S promoter and a kanamycin resistance marker. The vector is used for overexpression of the HsCIPK17 gene in rice. Using cDNA as a template, using HsCIPK17 specific primers (table 1) to amplify a corresponding HsCIPK17 target fragment, and using a PCR amplification system (50 μ L system) as follows: PCR H2O17.5. mu.L, 2 XPrimerSTAR GCBuffer 25. mu.L, PrimerSTAR HS DNA polymerase 0.5. mu.L, dNTP mix 4. mu.L, Primer F1. mu.L, Primer R1. mu.L, cDNA 1. mu.L. The PCR amplification procedure was as follows: pre-denaturation at 94 deg.C for 3min, denaturation at 98 deg.C for 10s, annealing at 65 deg.C for 5s, extension at 72 deg.C for 1min, 35 cycles, extension at 72 deg.C for 10min, and storage at 16 deg.C. Then, the objective fragment was ligated to the vector by molecular cloning procedures such as purification (gel recovery), digestion, ligation, etc., and the ligation product was transformed into competent cells of Escherichia coli JM109 by heat shock. PCR and enzyme digestion are utilized to carry out positive clone analysis and identification, then sequencing analysis is carried out, and finally the correct 35S-HsCIPK 17 overexpression vector is obtained. HsCIPK17 is shown in figure 1, and the identification result is shown in figure 2.
TABLE 1 primer sequences for cloning, RT-PCR and fluorescent quantitative PCR
Based on FIG. 2, it can be seen that the construction of the overexpression vector for HsCIPK17 was successful.
Example 4 cultivation of transgenic Rice overexpressing the HsCIPK17 Gene (FIG. 3 and Table 2)
The rice transgenic background material is japonica rice Nipponbare (Nipponbare). The transgenic method comprises the following steps:
(1) agrobacterium tumefaciens (EHA105) was transformed and the HsCIPK17 overexpression vector (35S:: HsCIPK17) was introduced into Agrobacterium tumefaciens competence (EHA105) by electroporation.
(2) Pre-culturing rice callus, taking a rice Nipponbare mature embryo as a test material to induce the callus, and basically comprising the following steps:
mechanically shelling mature rice seeds, and selecting full, smooth and plaque-free seeds;
② placing the seeds into a 250mL sterile triangle flask, and disinfecting for 2min by 70 percent alcohol;
③ washing with sterile water for 3-5 times, adding 30 percent NaClO solution, adding 1 drop of Tween-20 into every 50mL, and placing on a shaking table at 180rpm for sterilization for 30 min;
fourthly, repeating the previous step, and washing the seeds for 4 to 5 times by using sterile water (so that the seeds are operated on a clean bench);
pouring sterile water, placing the seeds on sterile filter paper to absorb residual water, then inoculating the sterilized seeds on an N6D culture medium, continuously culturing for 5-7 days at 32 ℃ under illumination, and inducing a large amount of callus (observing the callus induction condition of the seeds every 1-2 days in the culture process, simultaneously checking the pollution condition, and timely transferring the uncontaminated seeds into a clean N6D culture medium).
(3) Infection and removal of Agrobacterium
Firstly, shaking bacteria, taking agrobacterium EHA105 strain containing corresponding vectors to scratch on LB +50mg/L Rif +50mg/L Kan culture medium, and carrying out dark culture for 2 days at 28 ℃. Single colonies were picked in 5mL LB + Kan + Rif broth and shaken at 28 ℃ for about 24 h. 500uL of the bacterial liquid is taken to be placed in 50mL of AAM culture medium containing 10-20mg/L of AS, and cultured overnight at 220-250rpm until the bacteria is OD600About 0.1.
Secondly, infection, transferring the pre-cultured callus into a 250mL sterile triangular flask, then pouring 50mL agrobacterium suspension, and gently shaking for about 1.5 min; the callus was decanted and excess bacteria solution was aspirated through sterile filter paper and dried for 30 min.
③ Co-culture, the surface of the co-culture medium (N6D-AS, i.e., N6D medium containing 10-20mg/L AS) was covered with a layer of AAM-impregnated sterile filter paper, and the callus was inoculated on the filter paper and cultured in the dark at 25 ℃ for 3 days.
And fourthly, removing bacteria, transferring the callus into a sterile triangular flask after 3 days of culture, firstly washing the callus with sterile water for 4 to 5 times until the liquid is not turbid, then adding a proper amount of sterile water containing 500mg/L Cef to wash the callus, placing the callus on a shaking table at 28 ℃ and 120rpm, replacing the sterile water containing 500mg/L Cef at intervals of 30min, and finally washing the callus with the sterile water. After the bacteria is removed, putting the callus on filter paper for airing (2-3 h).
(4) Selection and differentiation of resistant callus, inoculating the callus after degerming on selection medium (N6D +150mg/L G418+500mg/L Cef), and culturing at 32 deg.C for two weeks under continuous illumination. The vigorous resistant callus is transferred to a differentiation medium (RE-III +0.02mg/L NAA +2mg/L KT +125mg/L Cef +100mg/L G418) to induce differentiation, and the green bud is grown after continuous illumination culture at 32 ℃.
(5) Rooting and hardening seedlings, wherein the differentiated seedlings need to be transplanted to a rooting medium (HF +125mg/L Cef +70mg/L G418) in time to induce rooting due to limited nutrition on the differentiation medium. After 2-3 weeks, the rooting bottle of the vigorous tissue culture seedling is opened, a proper amount of sterile water is added to isolate pathogens, the seedling is hardened for about three days, agar is eluted, and water culture is carried out.
Remarks explanation:
the regenerated test-tube seedling after hardening off is T1 generation, RT-PCR detects T1 generation plant, confirms that the transgenic plant containing 35S HsCIPK17 carrier is over-expression strain;
the T3 generation overexpression homozygote strain is obtained by G418-resistance selection (namely, separation conditions are not required to appear) to obtain a T3 generation homozygote strain of the HsCIPK17 overexpression transgenic strain.
Example 5 detection of expression level of target Gene in transgenic Rice
(1) RNA extraction and cDNA Synthesis
Total RNA was extracted from each strain of transgenic rice of the T1 generation according to the instructions of the RNA extraction Kit RNeasy Plant Mini Kit (QIAGEN), and the RNA concentration was determined using NanoDrop. cDNA synthesis was performed using the total RNA obtained above, reverse transcription was performed according to the synthesis method provided by the reverse transcription Kit ReverTra Ace qPCR RT Kit (TOYOBO), and the synthesis system (20. mu.L) was as follows: 5 × RT Buffer 4 μ L, RT Enzyme Mix 1 μ L, Primer Mix 1 μ L, RNA 5 μ L, nucleic-free water 9 μ L.
And (3) RNA denaturation, namely putting 0.1-1 mu g of RNA into a PCR tube, performing constant volume to 5 mu L by using a nucleic-free water, uniformly mixing, putting into a PCR instrument, performing denaturation at 65 ℃ for 5min, immediately taking out, inserting into ice, and rapidly cooling to prevent renaturation.
Reverse transcription program, reverse transcription 15min at 37 ℃; inactivating enzyme at 98 deg.C for 5 min; keeping the temperature at 16 ℃.
(2) Primers were designed for RT-PCR analysis using DNAStar Lasergene 7.1 software based on the CDS coding sequence of HsCIPK 17. Specific primer sequences (RT-PCR assay) are shown in Table 1.
(3) RT-PCR reaction parameters, cDNA is taken as a template, OsActin is taken as an internal reference, and the RT-PCR reaction system is as follows: PCR H2O8.2. mu.L, 2 XTaq Master Mix 10. mu.L, Primer F0.4. mu.L, Primer R0.4. mu.L, cDNA 1. mu.L. The RT-PCR reaction procedure was as follows: pre-denaturation at 94 deg.C for 3min, denaturation at 94 deg.C for 30s, annealing at 60 deg.C for 30s, extension at 72 deg.C for 1min, 27 cycles, extension at 72 deg.C for 5min, and storage at 16 deg.C.
The PCR products were detected by electrophoresis using 1% agarose gel, and the results are shown in FIG. 4.
As shown in fig. 4, the following conclusions can be drawn: the gene expression of the HsCIPK17 overexpression transgenic lines was improved compared to the non-transgenic controls, wherein the overexpression lines are shown in Table 2. Therefore, the transgenic rice obtains the expected transgenic effect.
Table 2, the obtained transgenic lines of T1 generation and overexpression homozygous lines of T3 generation
FIG. 4 shows the semi-quantitative RT-PCR assay, from which it can be seen that: 6. the expression of the strains 8 and 9 (namely the corresponding T1 generation numbers are T1.6, T1.8 and T1.9) is obviously stronger than that of the control NC wild type Nipponbare, which indicates that the constructed vector is successfully arranged in the strains; the T3 generation is based on the T1 generation, and corresponding homozygous strains are obtained by separation and selection, namely the corresponding T3 generation numbers are T3.6, T3.8 and T3.9.
From this table 2, it can be seen that: the transgenic rice obtains expected transgenic effect, obtains over-expression homozygous strain T3 generation, can perform experimental research on the resistance function of HsCIPK17, and ensures the stability of data.
Example 6 screening of transgenic homozygous lines of rice T3 Generation
After breeding the high-expression strain to T3 generation, screening the homozygous strain. About 40 seeds were selected from each plant and placed in a 37 ℃ biochemical incubator for germination. Soaking the seeds in 0.6% dilute nitric acid for 16h (promoting germination), then soaking the seeds in tap water for two days (changing water once every day), inserting the seeds into a solid culture medium of 0.6% Agar +20-80mg/L G418 after the seeds are exposed to white, and carrying out resistance screening. After two weeks of cultivation at 28 ℃ under 14h light/10 h dark, it was found that the growth conditions (aerial parts and roots) of the resistant seedlings and non-resistant seedlings were significantly different, and homozygote or heterozygote was determined according to the separation ratio. If all seedlings of the same plant have resistance to the selection medium and no segregation occurs (note: homozygous or not, and above all, no segregation occurs), this plant is an homozygous line. See fig. 5 and table 2.
From fig. 5, it can be seen that: the rice T3 generation transgenic homozygous strain is obtained successfully.
Description of the drawings: in order to ensure the reliability of the material and the stability of subsequent experimental data, the invention carries out resistance screening on the obtained high-expression strains, and finally obtains the over-expression homozygote strains as the material for subsequent functional verification. Seeds of T2 generations are harvested from T1 generations of plants, screened by a resistant medium containing 20mg/L G418, combined with a Mendelian segregation ratio, heterozygous lines of a 3:1 segregation ratio are selected, and propagated for one generation to obtain seeds of T3 generations. Homozygous line selection was performed on T3 seed on Agar medium containing 20-80mg/LG418G418 as shown in FIG. 5A. The resistant seedlings were able to grow normally in roots and leaves, while wild type seedlings were significantly inhibited in growth, the plants were dwarf, and the leaves were not spread, and root growth was also significantly inhibited (fig. 5B). If the selected line is a heterozygote, it will show a segregation ratio close to 3:1, i.e., 1/3 seedlings 'leaves and roots are inhibited from growing, while the rest 2/3 seedlings' leaves and roots grow normally; when the selected strain is homozygote, all the seedlings have normal leaf and root growth. By the method, the overexpression transgenic strains of the HsCIPK17 expression vector are screened into homozygote strains.
Example 7 determination of transcriptional expression of endogenous HsCIPK17 in annual wild barley induced by abiotic stress such as heavy metals
In order to detect the influence of abiotic stress such as heavy metal on transcription expression level of endogenous HsCIPK17 in the annual wild barley in Qinghai-Tibet plateau, RNeasy Plant Mini Kit (Qiagen) is used for separating and extracting total RNAs of all treated seedlings or roots.
The treatment was carried out in 20. mu.M HgCl2、20μM CdCl2、0.5mM K2Cr2O7400mM NaCl and 20. mu.M ABA, treated at increasing concentrations for 0, 1, 3, 6, 12 and 18 hours.
The First Strand of cDNA was synthesized using the SuperScript III First-Strand Synthesis System (Invitrogen). The qRT-PCR assay used a Thunderbird SYBR qPCR mix (Toyobo) and a StepOneNus Real-Time PCR System (Applied Biosystems). The reaction volume was 20. mu.l, containing 10. mu.l of 2 XSSYBR qPCR mix (Toyobo),10ng of cDNA, 1. mu.M of each gene-specific primer. And (3) PCR circulation: each cycle is at 95 ℃ for 3 min; 40 cycles 95 ℃ 5sec, 60 ℃ 50 sec. The resulting data were collected and analyzed using StepOne Software v 2.1. The reference gene HvActin was transcribed at the level (Table 2). The transcript levels of HsCIPK17 after various periods of stress treatment are expressed as relative values at the 0 time point (control, set at 1.0). Statistical analysis Using t-test (Student's t-test, two tests; type 2), the transcript levels of three independent experiments were compared to controls at different time points. See fig. 6.
From fig. 6, it can be seen that: abiotic stresses such as heavy metals and the like have obvious and obvious influence on the transcription and expression level of endogenous HsCIPK17 in the annual wild barley in the Qinghai-Tibet plateau.
Example 8 determination of root elongation of Rice T3 Generation against stress
The root Relative Elongations (RERs) were first used to determine the optimal treatment concentrations for various stress treatments. Culturing 8-10 seedlings of wild rice (Nipponbare) with root length of 3-4cm in water for 4 days, respectively, with treatment solution (containing 0.1mM CaCl) with different concentration gradients2) And blank control (Mock, 0.1mM CaCl)2) The treatment was carried out for 24 hours. Measuring the length of primary root before and after treatment, and calculating the relative elongation rate RER (%) ═ L of the primary rootT24-LT0)/(Lmock24-Lmock0) X 100% where LT0And LT24The length (mm) of primary roots before and after stress treatment, and Lmock0And Lmock24The length (mm) of the primary root before and after the blank control treatment. The treatment concentration when the relative elongation RER of the roots is close to 50% is determined as the optimum concentration. Finally, the optimal treatment concentration (containing 0.1mM CaCl) of each stress is determined2) Respectively as follows: 0.5. mu.M HgCl2，5μM CdCl2，5μM K2Cr2O750mM NaCl and 1. mu.M ABA. Transgenic and non-transgenic (NT) seedlings with root length of 3-4cm are stressed for 24 hours, the length of the primary root is measured before and after the stress treatment, and the relative elongation of the primary root of each transgenic line is calculated according to the formula. Three independent experiments, the data determined for each 45 seedlings treated, were used for statistical analysis, and Student's t-test (type 2) was evaluated statistically against the non-transgenic line (NT).
According to statistical analysis, the transgenic rice is proved to have remarkably enhanced capability of resisting abiotic stress such as heavy metal and the like and obvious abiotic stress characteristics such as heavy metal resistance and the like. See fig. 7.
Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Sequence listing
<110> Lanzhou university
<120> application of Qinghai-Tibet plateau wild barley HsCIPK17 in improvement of abiotic stress resistance/tolerance of rice
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gcgcggcacc gcgccaccgg ggaccacttc gccgtcaaga tcctcgaccg cggccgggtg 180
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aacatcaaga tctctgattt tggtctcagt gctttaccac aacatctcgg gaatgatgga 540
ttgctgcata caacctgtgg tagccccaac tatattgctc ctgaggttct gcagaacaga 600
ggttacgacg gatcattgtc ggatatctgg tcttgtggag taattcttta cataatgctc 660
gtaggaaacc ttccgtttga tgaccgaaat atggttgttc tttatcagaa gattttcaag 720
ggtgacgctc agatcccgga gtggctttct cccagtgcac aaaacctcct tcgtaggatt 780
cttgaaccaa atccgaggaa gaggattaac atggcagaga taaaaataca cgaatggttt 840
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gcaattctac ctatcaaaca gcaaattagt gaagcacccg gcgacaagag cactcatcag 960
atgaacgctt ttcagctgat cggaatggca tcctccctcg atctttcagg tttatttgag 1020
gaagagggag tgtcccagag aaagatcagg ttcacatcag cacaaccacc gaaggatttg 1080
ttcgacaaga ttgaagtgtc cgcgacacag tcggggttcc atgtccagag agcgcatagc 1140
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Claims (2)
1. The application of the calcineurin B protein interacting protein kinase gene HsCIPK17 of the annual wild barley (Hordeum spontanemum C. Koch) in Qinghai-Tibet plateau is characterized in that: HsCIPK17 is used for constructing transgenic rice (Oryza sativa L.), and the transgenic rice has heavy metal stress tolerance and also has salt tolerance and abscisic acid stress tolerance; the heavy metal is mercury, cadmium or chromium; can promote root elongation;
the nucleotide sequence of the gene HsCIPK17, GenBank accession number JN 655677.
2. The use of the wild barley gene HsCIPK17 as claimed in claim 1, wherein: the rice mature embryo callus is transformed by the HsCIPK17 gene, and then the transformed rice cells are cultivated into a transgenic plant which has 3 abiotic stress resistances of heavy metal, salt resistance and abscisic acid and can promote root elongation.
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